Zen 2009 light edition software

The 3D gels were washed with PBS(+) and collagens were mildly digested with collagenase 1 (Sigma) for about 10 min at 37 °C. After washing with PBS(+), cyst cells were fixed with 4% paraformaldehyde for 30 min. After washing 3 times with PBS(+), 3D gels were permeabilised for 30 min at room temperature with the permeabilisation solution: PBS(+) with 0.5% Triton X-100 (Sigma) and 5% donkey serum (Millipore). They were incubated with diluted primary antibodies overnight at 4 °C. The cells were washed with PBS(+)/0.5% Triton several times, and then incubated with diluted secondary antibodies for 40 minutes at room temperature. Antibodies were diluted with permeabilisation solution. Then the cells were washed with PBS(+)/0.5% Triton followed by a PBS(+) wash, and their nuclei were stained with 4′,6-diamidino-2phenylindole dihydrochloride (DAPI, Sigma). Primary and secondary antibodies are listed in Supplementary Table S2. Normal goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA), negative control mouse IgG1 (Dako, Glostrup, Denmark), and normal rabbit IgG (Santa Cruz Biotechnology) were used as negative controls for the appropriate antibodies. Cysts were imaged under confocal microscopy LSM700 and analysed by ZEN 2009 Light Edition software (Carl Zeiss, Jena, Germany).

Trophozoites were grown on coverslips, fixed with 4% paraformaldehyde at 37 °C for 1 h, permeabilized with 0.5% Triton X-100, and blocked with 10% fetal bovine serum in PBS. Then, cells were incubated at 4 °C overnight (ON) with either α-EhVps22 (1:100), α-EhVps25 (1:100), α-EhVps36 (1:100), α-EhVps23 (1:50), or α-EhVps20 (1:50) antibodies. After extensive washing, samples were incubated for 30 min at 37 °C with α-mouse pacific blue-labeled, α-rat TRITC-labeled, or α-rabbit FITC-labeled secondary antibodies (1:100). Nuclei were stained with 40′6-Diamidino-2- Phenylindole (DAPI). Fluorescence was preserved using VECTASHIELD antifade reagent (Vector), examined through a Carl Zeiss LMS 700 confocal microscope in laser sections of 0.5 µm, and processed with ZEN 2009 Light Edition Software (Zeiss, Dublin, CA, USA). We considered at least 25 confocal images using the ImageJ 1.45v software and JACoP plugin to evaluate the fluorescence intensity and co-localization between proteins.

All MDMs were cultivated on round coverslips within 12-well plates in the 5% CO₂ incubator at 37 ℃. The MDMs were infected with either BTV2 (MOI = 1) or BTV12 (MOI = 0.5) for 2 h. After 2 h incubation, the culture supernatants were replaced with fresh medium and harvested 12 hpi. To decrease the non-specific binding of antibodies with Fc receptors on MDMs, azide-free Fc receptor blocker (20 μL/10⁵ cells, Innovex Biosciences Inc., Richmond, CA, USA) was used for blocking for 30 min. After blocking, all cells were fixed with 4% paraformaldehyde (PFA, Merck, Darmstadt, Germany) in PBS for 3 min.
The IFA staining protocol was as described in the previous study [7 (link)]. Macrophage surface marker CD172a was labeled with the mouse anti-bovine CD172a antibody, with a secondary antibody of anti-mouse IgG1 conjugated with PerCP/Cyanine5.5 (Rat IgG, BioLegend, San Diego, CA, USA). BTV signals were labeled by rabbit anti-BTV-NS3 antibody (GenScript, Piscataway, NJ, USA), with a secondary antibody of goat anti-rabbit IgG conjugated with Alexfluor 546 (Thermo, Waltham, MA, USA). After staining, DAPI-Fluoromount-G Mounting Medium (Invitrogen, Carlsbad, CA, USA) was used to mount the slide. Cells were microphotographed with a Zeiss LSM 780 confocal microscope by using ZEN 2009 Light Edition software (Carl Zeiss, Oberkochen, Germany).

Immunofluorescence was performed to analyze the representative expression patterns of the epithelial cell phenotype (E-cadherin), the mesenchymal cell phenotype (α-SMA), and a stem cell marker (CXCR4). These cells were triple-stained with E-cadherin, α-SMA, and CXCR4. The stained slides were visualized with a LSM 510 META Laser Scanning Microscope (Carl Zeiss, Jena, Germany) and analyzed with the ZEN 2009 Light Edition software (Carl Zeiss, Jena, Germany).

To investigate localization of EhPKMT2, parasites were grown on coverslides and subsequently subjected to the treatments described above; then, trophozoites were fixed and permeabilized with cold methanol for 5 min. Non-specific binding sites were blocked with 10% FBS in PBS. Next, samples were incubated overnight at 4 °C with the anti-EhPKMT2 antibody (dilution 1:50) and later with an Alexa 488-conjugated secondary antibody (Invitrogen catalog. no. A-11008, Waltham, MA, USA) (1:400). Nuclei were counterstained with 4′,6-diamidino-2-Phenylindole (DAPI) and, finally, cells were observed through a confocal microscope (Carl Zeiss LSM 700, objective 40×, N.A. 1.3, laser 488 nm: 2.0%) and processed with ZEN 2009 Light Edition Software (Zeiss, Jena, Germany).